Abstract
Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer +/aerA+/hly +/ast +/alt + was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly + (85%) and aerA+ (78.7%), while in A. caviae complex strains was aerA+ (32.8%). All A. veronii complex sobria strains were aer +/aerA+. All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.
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Abbott SL, Cheung WK, Janda JM (2003) The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J Clin Microbiol 41:2348–2357. doi:10.1128/JCM.41.6.2348-2357.2003
Abrami L, Fivaz M, van der Goot FG (2000) Adventures of a pore-forming toxin at the target cell surface. Trends Microbiol 8:168–172. doi:10.1016/S0966-842X(00)01722-4
Abrami L, Fivaz M, Glauser PE, Sugimoto N, Zurzolo C, van der Goot FG (2003) Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin. Infect Immun 71:739–746. doi:10.1128/IAI.71.2.739-746.2003
Aguilera-Arreola MG, Hernandez-Rodriguez C, Zuniga G, Figueras MJ, Castro- Escarpulli G (2005) Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes. FEMS Microbiol Lett 242:231–240. doi:10.1016/j.femsle.2004.11.011
Aguilera-Arreola MG, Hernandez-Rodriguez C, Zuniga G, Figueras MJ, Garduño RA, Castro-Escarpulli G (2007) Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study. Can J Microbiol 53:877–887. doi:10.1139/W07-051
Al-Benwan K, Abbott S, Janda JM, Huys G, Albert MJ (2007) Cystitis caused by Aeromonas caviae. J Clin Microbiol 45:2348–2350. doi:10.1128/JCM.00480-07
Araújo VS, Pagliares VA, Queiroz MLP, Freitas-Almeida AC (2002) Occurrence of Staphylococcus and enteropathogens in soft cheese commercialized in the city of Rio de Janeiro, Brazil. J Appl Microbiol 92:1172–1177. doi:10.1046/j.1365-2672.2002.01656.x
Chopra AK, Houston CW, Peterson JW, Jin GF (1993) Cloning, expression, and sequence analysis of a cytolytic enterotoxin gene from Aeromonas hydrophila. Can J Microbiol 39:513–523
Couto CR, Oliveira SS, Queiroz ML, Freitas-Almeida AC (2007) Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells. Lett Appl Microbiol 45:405–410. doi:10.1111/j.1472-765X.2007.02199.x
Edberg SC, Browne FA, Allen MJ (2007) Issues for microbial regulation: Aeromonas as a model. Crit Rev Microbiol 33:89–100. doi:10.1080/10408410601172180
Fadl AA, Galindo CL, Sha J, Zhang F, Garner HR, Wang HQ, Chopra AK (2007) Global transcriptional responses of wild-type Aeromonas hydrophila and its virulence-deficient mutant in a murine model of infection. Microb Pathog 42:193–203. doi:10.1016/j.micpath.2007.02.002
Falcón R, Castro TLA, Luna MG, Freitas-Almeida AC, Yano T (2006) Detection of hemolysins in Aeromonas spp. isolates from food sources. PCR analysis and biological activity. In: Food-borne pathogens—methods and protocols, vol 21. Humana Press (ed), Totowa, NJ, pp 3–13
Figueras MJ, Aldea MJ, Fernandez N, Aspiroz C, Alperi A, Guarro J (2007) Aeromonas hemolytic uremic syndrome. A case and a review of the literature. Diagn Microbiol Infect Dis 58:231–234. doi:10.1016/j.diagmicrobio.2006.11.023
Freitas AC, Souza SMS, Macedo LC, Pinto EC, Pereira SS (1998) Aeromonas species associated with gastroenteritis in children: prevalence, characteristics and virulence properties. Rev Microbiol 29:152–157
Gonzalez-Serrano CJ, Santos JA, Garcia-Lopez ML, Otero A (2002) Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case. J Appl Microbiol 93:414–419. doi:10.1046/j.1365-2672.2002.01705.x
Granum PE, O’Sullivan K, Tomás Ormen O (1998) Possible virulence factors of Aeromonas spp. from food and water. FEMS Immunol Med Microbiol 21:131–137. doi:10.1111/j.1574-695X.1998.tb01158.x
Heuzenroeder MW, Wong CY, Flower RL (1999) Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model. FEMS Microbiol Lett 174:131–136. doi:10.1111/j.1574-6968.1999.tb13559.x
Hofer E, Reis CM, Theophilo GN, Cavalcanti VO, Lima NV, Henriques Mde F (2006) Aeromonas associated with an acute diarrhea outbreak in Sao Bento do Una, Pernambuco. Rev Soc Bras Med Trop 39:217–220
Huang LJ, Chen HP, Chen TL, Siu LK, Fung CP, Lee FY, Liu CY (2006) Secondary Aeromonas peritonitis is associated with polymicrobial ascites culture and absence of liver cirrhosis compared to primary Aeromonas peritonitis. APMIS 114:772–778. doi:10.1111/j.1600-0463.2006.apm_470.x
Isonhood JH, Drake M (2002) Aeromonas species in foods. J Food Prot 65:575–582
Janda JM, Abbott SL (1998) Evolving concepts regarding the genus Aeromonas: an expanding Panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 27:332–344. doi:10.1086/514652
Joseph SW, Carnahan AM (2000) Update on the genus Aeromonas. Aeromonas ASM News 66:218–223
Karunakaran T, Devi BG (1994a) Characterisation of haemolytic activity from Aeromonas caviae. Epidemiol Infect 112:291–298
Karunakaran T, Devi BG (1994b) Factors influencing beta-galactosidase activity of Aeromonas caviae. J Basic Microbiol 34:245–252. doi:10.1002/jobm.3620340407
Khan AA, Kim E, Cerniglia CE (1998) Molecular cloning, nucleotide sequence, and expression in Escherichia coli of a hemolytic toxin (aerolysin) gene from Aeromonas trota. Appl Environ Microbiol 64:2473–2478
Kingombe CI, Huys G, Tonolla M, Albert MJ, Swings J, Peduzzi R, Jemmi T (1999) PCR detection, characterization, and distribution of virulence genes in Aeromonas spp. Appl Environ Microbiol 65:5293–5302
Maas R (1983) An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid 10(3):296–298. doi:10.1016/0147-619X(83)90045-8
Mok MY, Wong SY, Chan TM, Tang WM, Wong WS, Lau CS (2006) Necrotizing fasciitis in rheumatic diseases. Lupus 15:380–383. doi:10.1191/0961203306lu2314cr
Sen K, Rodgers M (2004) Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification. J Appl Microbiol 97:1077–1086. doi:10.1111/j.1365-2672.2004.02398.x
Sha J, Kozlova EV, Chopra AK (2002) Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect Immun 70:1924–1935. doi:10.1128/IAI.70.4.1924-1935.2002
Sha J, Kozlova EV, Fadl AA, Olano JP, Houston CW, Peterson JW, Chopra AK (2004) Molecular characterization of a glucose-inhibited division gene, gidA, that regulates cytotoxic enterotoxin of Aeromonas hydrophila. Infect Immun 72:1084–1095. doi:10.1128/IAI.72.2.1084-1095.2004
Sha J, Pillai L, Fadl AA, Galindo CL, Erova TE, Chopra AK (2005) The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila. Infect Immun 73:6446–6457. doi:10.1128/IAI.73.10.6446-6457.2005
Shaw J, Thornley J, Eley A (1997) Adherence and invasion of Aeromonas caviae to monolayer cells. Adherence and invasion of Aeromonas caviae. Adv Exp Med Biol 412:217–219
Sinha S, Shimada T, Ramamurthy T, Bhattacharya SK, Yamasaki S, Takeda Y, Nair GB (2004) Prevalence, serotype distribution, antibiotic susceptibility and genetic profiles of mesophilic Aeromonas species isolated from hospitalized diarrhoeal cases in Kolkata, India. J Med Microbiol 53:527–534. doi:10.1099/jmm.0.05269-0
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882. doi:10.1093/nar/25.24.4876
Trajano RS, Pinto EC, Reis LS, Oliveira SS, Vanderweerelt M, Nunez MLS, Taketani RG, Araújo FV, Rosado AS, Freitas-Almeida AC (2003) Detecção de cepas multi-resistentes de Aeromonas spp. e análise do índice de poluição fecal de águas superficiais da Lagoa Rodrigo de Freitas, Rio de Janeiro, Brasil. 22º Congresso Brasileiro de Microbiologia Florianópolis (Santa Catarina), Brasil
von Graevenitz A (2007) The role of Aeromonas in diarrhea: a review. Infection 35:59–64. doi:10.1007/s15010-007-6243-4
Wang G, Tyler KD, Munro CK, Johnson WM (1996) Characterization of cytotoxic, hemolytic Aeromonas caviae clinical isolates and their identification by determining presence of a unique hemolysin gene. J Clin Microbiol 34:3203–3205
Wang G, Clark CG, Liu C, Pucknell C, Munro CK, Kruk TM, Caldeira R, Woodward DL, Rodgers FG (2003) Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR. J Clin Microbiol 41:1048–1054. doi:10.1128/JCM.41.3.1048-1054.2003
Acknowledgments
This work was supported by grants from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasil. The authors thank M. S. Esther Helena Rondon Prado and Gabriela B. Alves for technical assistance.
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Castilho, M.C.B., Castro, T.L.A., Araújo, V.S. et al. High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil. Antonie van Leeuwenhoek 96, 53–61 (2009). https://doi.org/10.1007/s10482-009-9335-6
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DOI: https://doi.org/10.1007/s10482-009-9335-6